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Literature summary extracted from

  • Kitanishi, K.; Cracan, V.; Banerjee, R.
    Engineered and native coenzyme B12-dependent isovaleryl-CoA/pivalyl-CoA mutase (2015), J. Biol. Chem., 290, 20466-20476 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
5.4.99.13 synthesis IcmF is potentially useful as a reagent for bioremediation of pivalic acid found in pharmaceutical wastewaters (pivalic acid esters are used as pro-drugs) and/or for the biosynthesis of a simple beta-nonfunctional alpha-quaternary carboxylic acid. Another potential application of IcmF is in metabolic engineering of pathways to produce branched C4 and C5 building blocks that can subsequently be converted to useful derivatives, for example, the corresponding isobutyl and pivalyl alcohols Cupriavidus metallidurans

Cloned(Commentary)

EC Number Cloned (Comment) Organism
5.4.99.13 gene icmF, recombinant expression of soluble His-MBP-tagged wild-type enzyme and His6-SUMO-tagged mutant enzyme in Escherichia coli strain BL21(DE3) Cupriavidus metallidurans

Protein Variants

EC Number Protein Variants Comment Organism
5.4.99.13 F598A site-directed mutagenesis, the mutation causes a 17fold increase in catalytic efficiency in pivalyl-CoA mutase activity and a concomitant about 240fold decrease in isobutyryl-CoA mutase activity compared to wild-type IcmF. The mutation of the single residue in IcmF tunes the substrate specificity to an about 4000fold increase in the specificity for the unnatural substrate. The F598A mutant is more susceptible to inactivation than wild-type IcmF Cupriavidus metallidurans
5.4.99.13 F598G site-directed mutagenesis, inactive mutant Cupriavidus metallidurans
5.4.99.13 F598I site-directed mutagenesis, the isobutyryl-CoA mutase activity of the mutant is diminished and the isovaleryl-CoA mutase activity is increased compared to wild-type Cupriavidus metallidurans
5.4.99.13 F598L site-directed mutagenesis, the isobutyryl-CoA mutase activity of the mutant is diminished and the isovaleryl-CoA mutase activity is increased compared to wild-type Cupriavidus metallidurans
5.4.99.13 F598V site-directed mutagenesis, the isobutyryl-CoA mutase activity of the mutant is diminished and the isovaleryl-CoA mutase activity is increased compared to wild-type Cupriavidus metallidurans
5.4.99.13 additional information AdoCbl is quantitatively converted to cob(II)alamin when isovaleryl-CoA is added to F598A or to the F598I/L or Q742A mutants inactivating the enzyme, The F598A mutant inactivates 3fold more rapidly than wild-type IcmF Cupriavidus metallidurans
5.4.99.13 additional information engineering of enzyme IcmF to increase pivalyl-CoA mutase activityby targeting two active site residues predicted to be key to substrate selectivity based on sequence alignments and crystal structures. PCM-F is an artificially engineered variant of PCM (IcmF) in which the large and small subunits are fused via an 11-amino acid linker Cupriavidus metallidurans
5.4.99.13 Q742A site-directed mutagenesis, the mutant shows undetectable isovaleryl-CoA mutase activity and significantly diminished isobutyryl-CoA mutase activity compared to wild-type Cupriavidus metallidurans
5.4.99.13 Q742L site-directed mutagenesis, inactive mutant Cupriavidus metallidurans
5.4.99.13 Q742N site-directed mutagenesis, the mutant shows undetectable isovaleryl-CoA mutase activity and significantly diminished isobutyryl-CoA mutase activity compared to wild-type Cupriavidus metallidurans

Inhibitors

EC Number Inhibitors Comment Organism Structure
5.4.99.13 additional information inactivation rates of OH2Cbl formation for wild-type and mutant enzymes, overview Cupriavidus metallidurans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.4.99.13 additional information
-
 additional information the pivalyl-CoA mutase activity of engineered PCM shows a sigmoidal dependence on the concentration of isolvaleryl-CoA Cupriavidus metallidurans
5.4.99.13 0.2
-
 isovaleryl-CoA recombinant engineered PCM-F mutant, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.36
-
 2-methylpropanoyl-CoA recombinant mutant F598I, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.37
-
 isovaleryl-CoA recombinant engineered PCM mutant, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.45
-
 2-methylpropanoyl-CoA recombinant mutant F598A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.51
-
 isovaleryl-CoA recombinant mutant F598A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.56
-
 isovaleryl-CoA recombinant mutant F598L, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.58
-
 isovaleryl-CoA recombinant mutant F598I, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.62
-
 isovaleryl-CoA recombinant mutant F598V, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.67
-
 2-methylpropanoyl-CoA recombinant mutant F598V, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.79
-
 2-methylpropanoyl-CoA recombinant mutant Q742A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.89
-
 2-methylpropanoyl-CoA recombinant mutant F598L, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 1.1
-
 2-methylpropanoyl-CoA recombinant wild-type enzyme, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 1.4
-
 isovaleryl-CoA recombinant wild-type enzyme, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 2.4
-
 2-methylpropanoyl-CoA recombinant mutant Q742N, pH 7.5, 37°C Cupriavidus metallidurans

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
5.4.99.13 additional information
-
 size exclusion chromatography yields an estimated molecular mass of 135 kDa, consistent with the large subunit being a homodimer. The recombinant small subunit of PCM is purified as N-terminally His6-tagged protein and elutes with an apparent mass of 20 kDa in gel filtration, suggesting that it exists as a monomer based on the predicted mass of the polypeptide of 17.7 kDa. Gel filtration of a 1:1 mixture of the large and small subunits ofthe enzyme in presence of 5'-deoxyadenosylcobalamin, shows no evidence of complex formation, indicating weak interaction between the subunits under these conditions Cupriavidus metallidurans
5.4.99.13 20000
-
 recombinant detagged enzyme, small subunit monomer, gel filtration Cupriavidus metallidurans
5.4.99.13 135000
-
 recombinant detagged enzyme, large subunit dimer, gel filtration Cupriavidus metallidurans

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.4.99.13 2-methylpropanoyl-CoA Cupriavidus metallidurans interconversion butanoyl-CoA
-
 r
5.4.99.13 2-methylpropanoyl-CoA Cupriavidus metallidurans DSM 2839 interconversion butanoyl-CoA
-
 r

Organism

EC Number Organism UniProt Comment Textmining
5.4.99.13 Cupriavidus metallidurans Q1LRY0
-
-
5.4.99.13 Cupriavidus metallidurans DSM 2839 Q1LRY0
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
5.4.99.13 recombinant His-MBP-tagged wild-type enzyme and His6-SUMO-tagged mutant enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, tag cleavage by TEV protease, dialysis, amylose affinity chromatography (only wild-type), and gel filtration Cupriavidus metallidurans

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
5.4.99.13 0.18
-
 purified recombinant enzyme, pivalyl-CoA mutase activity of engineered PCM, pH 7.5, 37°C Cupriavidus metallidurans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.4.99.13 2-methylpropanoyl-CoA interconversion Cupriavidus metallidurans butanoyl-CoA
-
 r
5.4.99.13 2-methylpropanoyl-CoA interconversion, preferred substrate Cupriavidus metallidurans butanoyl-CoA
-
 r
5.4.99.13 2-methylpropanoyl-CoA interconversion Cupriavidus metallidurans DSM 2839 butanoyl-CoA
-
 r
5.4.99.13 2-methylpropanoyl-CoA interconversion, preferred substrate Cupriavidus metallidurans DSM 2839 butanoyl-CoA
-
 r
5.4.99.13 isovaleryl-CoA
-
 Cupriavidus metallidurans pivalyl-CoA
-
 r
5.4.99.13 isovaleryl-CoA
-
 Cupriavidus metallidurans DSM 2839 pivalyl-CoA
-
 r

Subunits

EC Number Subunits Comment Organism
5.4.99.13 homodimer 2 * 62300, recombinant detagged enzyme large subunit, SDS-PAGE Cupriavidus metallidurans
5.4.99.13 monomer 1 * 17700, about, recombinant detagged enzyme small subunit, sequence calculation Cupriavidus metallidurans
5.4.99.13 More IcmF is a natural variant in which the small and large subunits found in ICM are fused via a middle G-protein chaperone domain, domain structure and sequence comparisons, overview Cupriavidus metallidurans

Synonyms

EC Number Synonyms Comment Organism
5.4.99.13 AdoCbl-dependent PCM
-
 Cupriavidus metallidurans
5.4.99.13 CmIcmF
-
 Cupriavidus metallidurans
5.4.99.13 ICM
-
 Cupriavidus metallidurans
5.4.99.13 IcmF
-
 Cupriavidus metallidurans
5.4.99.13 isovaleryl-CoA/pivalyl-CoA mutase
-
 Cupriavidus metallidurans
5.4.99.13 PCM
-
 Cupriavidus metallidurans
5.4.99.13 pivalyl-CoA mutase
-
 Cupriavidus metallidurans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.4.99.13 37
-
 assay at Cupriavidus metallidurans

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.4.99.13 0.0036
-
 isovaleryl-CoA recombinant mutant F598I, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.0084
-
 isovaleryl-CoA recombinant mutant F598L, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.014
-
 isovaleryl-CoA recombinant engineered PCM mutant, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.015
-
 isovaleryl-CoA recombinant mutant F598V, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.022
-
 isovaleryl-CoA recombinant mutant F598A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.023
-
 isovaleryl-CoA recombinant engineered PCM-F mutant, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.062
-
 isovaleryl-CoA recombinant wild-type enzyme, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.08
-
 2-methylpropanoyl-CoA recombinant mutant F598A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.08
-
 2-methylpropanoyl-CoA recombinant mutant Q742A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.14
-
 2-methylpropanoyl-CoA recombinant mutant F598I, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.2
-
 2-methylpropanoyl-CoA recombinant mutant F598V, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 1.27
-
 2-methylpropanoyl-CoA recombinant mutant F598L, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 1.83
-
 2-methylpropanoyl-CoA recombinant mutant Q742N, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 48.33
-
 2-methylpropanoyl-CoA recombinant wild-type enzyme, pH 7.5, 37°C Cupriavidus metallidurans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.4.99.13 7.5
-
 assay at Cupriavidus metallidurans

Cofactor

EC Number Cofactor Comment Organism Structure
5.4.99.13 5'-deoxyadenosylcobalamin AdoCbl, required, the active sites in the CmIcmF dimer exhibit identical affinity for AdoCbl. Addition of isovaleryl-CoA to wild-type holo-IcmF results in formation of the intermediate cob(II)alamin. AdoCbl is quantitatively converted to cob(II)alamin when isovaleryl-CoA is added to F598A or to the F598I/L or Q742A mutants inactivating the enzyme. Cob(II)alamin formation in activates the enzyme Cupriavidus metallidurans

General Information

EC Number General Information Comment Organism
5.4.99.13 evolution enzyme IcmF belongs to the family of adenosylcobalamin-dependent isomerases, whose members catalyze carbon skeleton rearrangements using radical chemistry Cupriavidus metallidurans
5.4.99.13 additional information IcmF is a natural variant in which the small and large subunits found in ICM are fused via a middle G-protein chaperone domain, active site sequence comparisons, overview Cupriavidus metallidurans
5.4.99.13 physiological function the isobutyryl-CoA mutase variant, IcmF, catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA and it also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation Cupriavidus metallidurans

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.4.99.13 0.0062
-
 isovaleryl-CoA recombinant mutant F598I, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.015
-
 isovaleryl-CoA recombinant mutant F598L, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.0242
-
 isovaleryl-CoA recombinant mutant F598V, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.0378
-
 isovaleryl-CoA recombinant engineered PCM mutant, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.0431
-
 isovaleryl-CoA recombinant mutant F598A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.0443
-
 isovaleryl-CoA recombinant wild-type enzyme, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.101
-
 2-methylpropanoyl-CoA recombinant mutant Q742A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.115
-
 isovaleryl-CoA recombinant engineered PCM-F mutant, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.18
-
 2-methylpropanoyl-CoA recombinant mutant F598A, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.299
-
 2-methylpropanoyl-CoA recombinant mutant F598V, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.39
-
 2-methylpropanoyl-CoA recombinant mutant F598I, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 0.76
-
 2-methylpropanoyl-CoA recombinant mutant Q742N, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 1.43
-
 2-methylpropanoyl-CoA recombinant mutant F598L, pH 7.5, 37°C Cupriavidus metallidurans
5.4.99.13 43.94
-
 2-methylpropanoyl-CoA recombinant wild-type enzyme, pH 7.5, 37°C Cupriavidus metallidurans